Rodríguez-Herva J J, Reniero D, Galli E, Ramos J L
Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain.
Environ Microbiol. 1999 Dec;1(6):479-88. doi: 10.1046/j.1462-2920.1999.00058.x.
To generate mutants with altered lipopolysaccharides (LPS) of the wild-type Pseudomonas putida KT2442, we used the mini-Tn5luxAB-Km transposon. A mutant was found among luminescent colonies and selected as a negative clone in enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody (mAb) 7.3B, which recognizes the O-antigen of P. putida LPS. The DNA region of the LPS mutant interrupted by the minitransposon insertion was cloned and sequenced. Comparison of the deduced amino acid sequence with protein sequence databases showed similarity to the O-antigen polymerase (Wzy) of Salmonella enterica (muenchen). The wild-type gene was rescued by polymerase chain reaction (PCR), cloned into a broad-host-range plasmid and used to carry out complementation assays. The cloned gene was able to restore the wild-type phenotype of the P. putida wzy mutant. We constructed an isogenic mutant of the luminescent wzy mutant to which an oprL mutation was transferred by homologous recombination with an oprL::xylE cassette. The wzy mutants of P. putida were more sensitive to SDS, deoxycholate and EDTA than the corresponding parental strains. We analysed the ability of wzy, oprL and wzy oprL mutants of P. putida to colonize soil. In comparison with the wild-type strain, the ability of single mutants to colonize soil decreased; this characteristic was more evident for the double mutant, especially at high temperatures.
为了产生野生型恶臭假单胞菌KT2442脂多糖（LPS）改变的突变体，我们使用了mini-Tn5luxAB-Km转座子。在发光菌落中发现了一个突变体，并在使用识别恶臭假单胞菌LPS的O抗原的单克隆抗体（mAb）7.3B进行的酶联免疫吸附测定（ELISA）中被选为阴性克隆。克隆并测序了被mini转座子插入中断的LPS突变体的DNA区域。将推导的氨基酸序列与蛋白质序列数据库进行比较，发现与肠炎沙门氏菌（慕尼黑）的O抗原聚合酶（Wzy）相似。通过聚合酶链反应（PCR）拯救野生型基因，将其克隆到广宿主范围质粒中并用于进行互补试验。克隆的基因能够恢复恶臭假单胞菌wzy突变体的野生型表型。我们构建了一个发光wzy突变体的同基因突变体，通过与oprL::xylE盒的同源重组将oprL突变转移到该突变体中。恶臭假单胞菌的wzy突变体比相应的亲本菌株对SDS、脱氧胆酸盐和EDTA更敏感。我们分析了恶臭假单胞菌的wzy、oprL和wzy oprL突变体在土壤中定殖的能力。与野生型菌株相比，单突变体在土壤中定殖的能力下降；这种特性在双突变体中更明显，尤其是在高温下。
